1. Field of the Invention
The present invention relates to a method and apparatus for separating first particles from second particles. The invention has particular advantages in connection with separating stem cells, tumor cells, T cells, and red blood cells.
2. Description of the Related Art
In many different fields, liquids carrying particle substances must be filtered or processed to obtain either a purified liquid or purified particle end product. In its broadest sense, a filter is any device capable of removing or separating particles from a substance. Thus, the term "filter" as used herein is not limited to a porous media material but includes many different types of processes where particles are either separated from one another or from liquid.
In the medical field, it is often necessary to filter blood. Whole blood consists of various liquid constituents and particle constituents. Sometimes, the particle constituents are referred to as "formed elements". The liquid portion of blood is largely made up of plasma, and the particle constituents include red blood cells (erythrocytes), white blood cells (including leukocytes), and platelets (thrombocytes). While these constituents have similar densities, their average density relationship, in order of decreasing density, is as follows: red blood cells, white blood cells, platelets, and plasma. In addition, the particle constituents are related according to size, in order of decreasing size, as follows: white blood cells, red blood cells, and platelets. The size of red blood cells, however, may vary because red blood cells osmotically change size depending on the hypotonicity or hypertonicity of a liquid, such as plasma, in which the red blood cells are disbursed. When hypotonicity of plasma increases, the red blood cells osmotically become larger. Conversely, when hypertonicity of plasma increases, the red blood cells osmotically become smaller. Most current purification devices rely on density and size differences or surface chemistry characteristics to separate and/or filter the blood components.
Numerous therapeutic treatments require groups of particles to be removed from whole blood before either liquid or particle components can be infused into a patient. For example, cancer patients often require platelet transfusions after undergoing ablative, chemical, or radiation therapy. In this procedure, donated whole blood is processed to remove platelets and these platelets are then infused into the patient. However, if a patient receives an excessive number of foreign white blood cells as contamination in a platelet transfusion, the patient's body may reject the platelet transfusion, leading to a host of serious health risks.
Typically, donated platelets are separated or harvested from other blood components using a centrifuge. The centrifuge rotates a blood reservoir to separate components within the reservoir using centrifugal force. In use, blood enters the reservoir while it is rotating at a very rapid speed and centrifugal force stratifies the blood components, so that particular components may be separately removed. Centrifuges are effective at separating platelets from whole blood, however they typically are unable to separate all of the white blood cells from the platelets. Historically, blood separation and centrifugation devices are typically unable to consistently (99% of the time) produce platelet product that meets the "leukopoor" standard of less than 5.times.10.sup.6 white blood cells for at least 3.times.10.sup.11 platelets collected.
Because typical centrifuge platelet collection processes are unable to consistently and satisfactorily separate white blood cells from platelets, other processes have been added to improve results. In one procedure, after centrifuging, platelets are passed through a porous woven or non-woven media filter, which may have a modified surface, to remove white blood cells. However, use of the porous filter introduces its own set of problems. Conventional porous filters may be inefficient because they may permanently remove or trap approximately 5-20% of the platelets. These conventional filters may also reduce "platelet viability," meaning that once passed through a filter a percentage of the platelets cease to function properly and may be partially or fully activated. In addition, porous filters may cause the release of brandykinin, which may lead to hypotensive episodes in a patient. Porous filters are also expensive and often require additional time consuming manual labor to perform a filtration process.
Although porous filters are effective in removing a substantial number of white blood cells, they have drawbacks. For example, after centrifuging and before porous filtering, a period of time must pass to give activated platelets time to transform to a deactivated state. Otherwise, the activated platelets are likely to clog the filter. Therefore, the use of porous filters is not feasible in on-line processes.
Another separation process is one known as centrifugal elutriation. This process separates cells suspended in a liquid medium without the use of a membrane filter. In one common form of elutriation, a cell batch is introduced into a flow of liquid elutriation buffer. This liquid which carries the cell batch in suspension, is then introduced into a funnel-shaped chamber located in a spinning centrifuge. As additional liquid buffer solution flows through the chamber, the liquid sweeps smaller sized, slower-sedimenting cells toward an elutriation boundary within the chamber, while larger, faster-sedimenting cells migrate to an area of the chamber having the greatest centrifugal force.
When the centrifugal force and force generated by the fluid flow are balanced, the fluid flow is increased to force slower-sedimenting cells from an exit port in the chamber, while faster-sedimenting cells are retained in the chamber. If fluid flow through the chamber is increased, progressively larger, faster-sedimenting cells may be removed from the chamber.
Thus, centrifugal elutriation separates particles having different sedimentation velocities. Stoke's law describes sedimentation velocity (SV) of a spherical particle as follows: ##EQU1## where, r is the radius of the particle, .rho..sub.p is the density of the particle, .rho..sub.m is the density of the liquid medium, .eta. is the viscosity of the medium, and g is the gravitational or centrifugal acceleration. Because the radius of a particle is raised to the second power in the Stoke's equation and the density of the particle is not, the size of a cell, rather than its density, greatly influences its sedimentation rate. This explains why larger particles generally remain in a chamber during centrifugal elutriation, while smaller particles are released, if the particles have similar densities.
As described in U.S. Pat. No. 3,825,175 to Sartory, centrifugal elutriation has a number of limitations. In most of these processes, particles must be introduced within a flow of fluid medium in separate discontinuous batches to allow for sufficient particle separation. Thus, some elutriation processes only permit separation in particle batches and require an additional fluid medium to transport particles. In addition, flow forces must be precisely balanced against centrifugal force to allow for proper particle segregation.
Further, a Coriolis jetting effect takes place when particles flow into an elutriation chamber from a high centrifugal field toward a lower centrifugal field. The fluid and particles turbulently collide with an inner wall of the chamber facing the rotational direction of the centrifuge. This phenomenon mixes particles within the chamber and reduces the effectiveness of the separation process. Further, Coriolis jetting shunts flow along the inner wall from the inlet directly to the outlet. Thus, particles pass around the elutriative field to contaminate the end product.
Particle mixing by particle density inversion is an additional problem encountered in some prior elutriation processes. Fluid flowing within the elutriation chamber has a decreasing velocity as it flows in the centripetal direction from an entrance port toward an increased cross sectional portion of the chamber. Because particles tend to concentrate within a flowing liquid in areas of lower flow velocity, rather than in areas of high flow velocity, the particles concentrate near the increased cross-sectional area of the chamber. Correspondingly, since flow velocity is greatest adjacent the entrance port, the particle concentration is reduced in this area. Density inversion of particles takes place when the centrifugal force urges the particles from the high particle concentration at the portion of increased cross-section toward the entrance port. This particle turnover reduces the effectiveness of particle separation by elutriation.
In addition to red and white blood cells, plasma, and platelets, human blood also includes other particle components, such as T cells, stem cells, and, in some cases, tumor cells. These cells have substantially similar densities, but different sedimentation velocities and sizes. Generally, stem cells are larger than T cells and smaller than tumor cells. Some tumor cells (approximately 30%), however, are smaller than stem cells.
Existing purification devices are capable of purifying peripheral blood to isolate what is known as a peripheral cell collection for transfusion or reinfusion purposes. The peripheral cell collection typically includes primarily plasma, red blood cells, stem cells, and T cells, and may also include tumor cells if the donor's blood included such cells. Although transfusion of a peripheral cell collection is a common medical treatment, transfusion of a large number of T cells or tumor cells into a patient may have adverse consequences. Removal of T cells and tumor cells from a peripheral cell collection or whole blood before transfusion, however, is extremely difficult.
After undergoing a therapeutic treatment, such as chemotherapy or radiation therapy to eliminate cancerous tumor cells, cancer patients often receive a peripheral cell or bone marrow transfusion to replace stem cells destroyed as a side effect of the treatment. To reduce risks associated with infusing blood components from a foreign donor, some of these transfusions are autologous, where blood components collected from the patient are later reinfused back to the patient.
Blood components initially collected from cancer patients may include cancerous tumor cells, which are then infused back into the cancer patient during reinfusion. This reinfusion of tumor cells may diminish the effectiveness of therapeutic treatments aimed at reducing cancerous tumors in a patient's body.
Another type of transfusion, known as allogenic transfusion, involves collecting blood components from a donor and then infusing the collected blood components into a recipient who is different from the donor. Sometimes, however, the recipient of an allogenic transfusion experiences a disease commonly known as graft versus host disease. In graft versus host disease, T cells, which may accompany the blood components, are infused into the recipient and "recognize" that the recipient's body is "foreign" from that of the donor's. These T cells "attack" healthy cells in the recipient's body, rather than performing a normal immunological protective function.
Prior attempts to separate stem cells from tumor cells or separate stem cells from T cells prior to reinfusion have had limited success. In one separation method, a selective antibody is introduced into a collected blood component sample. The selective antibody chemically attaches to stem cells in the collection to "mark" them. To separate the marked stem cells from the remaining blood components, the collected blood components are passed between stationary beads coated with a material which chemically bonds with the selective antibody. These chemical bonds retain the marked stem cells on the beads to filter them from the remaining blood components. To remove the marked stem cells from the beads, a chemical solution is flushed through the beads to break the chemical bonds between the material and selective antibody. This separation process, however, is extremely expensive, tedious, and time consuming.
Centrifugal elutriation has been used to separate tumor cells from stem cells or to separate T cells from stem cells. With existing elutriation devices, however, this is time consuming, difficult, and of limited effectiveness.
For these and other reasons, there is a need to improve particle separation.